Nicotinamide Reduces the Infarct Volume in a Rat Model of Transient Middle Cerebral Artery Occlusion

BACKGROUND: Cerebral ischemia depletes ATP and causes irreversible tissue injury. Nicotinamide is a precursor of NAD+ and it is also a poly (ADP-ribose) polymerase (PARP) inhibitor that increases the neuronal ATP concentration and so protects against stroke. Therefore we examined whether nicotinamide could protect against cerebral ischemia by using a model of transient middle cerebral artery occlusion (MCAO) (reperfusion 2 h post ischemia) in Sprague-Dawley rats. METHODS: Nicotinamide (500 mg/kg) or normal saline was administered intraperitoneally 24 and 0 h before and after MCAO, respectively. The infarction volumes were determined with triphenyltetrazolium chloride staining 24 h after reperfusion. The nitrotyrosine, PAR polymer and PARP-1 expressions were examined by immunohistochemistry with using brain slices obtained from the rats that were sacrificed at 0, 15, 30, 60 and 120 min after reperfusion. RESULTS: The infarction volumes were significantly attenuated (21.8%, p<0.05). The nitrotyrosine expressions were increased at 0, 15 and 30 min, and those expressions for PARP polymer and PARP-1 were increased at 60 and 120 min, respectively. Nicotinamide partly reduced the expressions for nitrotyrosine and PAR polymer except for PARP-1. CONCLUSIONS: These results suggest that nicotinamide may attenuate ischemic brain injury through its antioxidant activity and the inhibition of PARP-1.

Minocycline Attenuates the Development of Allodynia: An Immunohistochemical Study on CD11b, GFAP and c-Fos in the Spinal Dorsal Horn in SD Rat

BACKGROUND: Minocycline, a semisynthetic second-generation tetracycline, is an antibiotic that has excellent ability to penetration into the CNS via the brain-blood barrier. Minocycline has emerged as a potent inhibitor of microglial activation, and it is an effective neuroprotective agent in experimental brain ischemia. Glial cell activation and proliferation are known to be associated with neuropathic pain in the peripheral nerve injuries. METHODS: The fifty percent threshold of withdrawal responses was measured in the hindpaws of SD rats following tight ligation of left fifth lumbar spinal nerve. Rats were sacrificed at 1, 3, 5, and 7 days and at 0.5, 1, 2, and 4 h post ligation (n=5/group/time point). Immunohistochemistry for GFAP, CD11b and c-Fos was done on the spinal cord at the level of the fifth lumbar nerve. Minocycline (45 mg/kg) and normal saline (300-400 microL) were administered intraperitoneally, 1 day and 1 h before the operations, and every day postoperatively until the rats were sacrificed. RESULTS: Treatment with minocycline reduced allodynia and the expressions of CD11b at 5 days and c-Fos at 1 and 2 h post operation compared with the saline treatment (control). CONCLUSIONS: It was thought that minocycline reduced the allodynia induced by tight ligation of the fifth lumbar spinal nerve in rats through the inhibition of microglial activation and c-Fos expression.

The Expression of Bcl-2, Bax, Cytochrome C and Caspase-3 in Camptothecin-Induced Apoptosis of Mouse 3T3 Fibroblasts

BACKGROUND: Camptothecin (CPT), which has been used for cancer treatment and apoptosis as an inhibitor of DNA topoisomerase I. We investigated the possibility that camptothecin induces anti-appoptotic bcl-2 and pro-apoptotic bax, cytochrome c and caspase-3. METHODS: We performed immunocytochemical stains for bcl-2, bax and cytochrome c, and also performed westem blots for caspase-3 and the three proteins above using mouse 3T3 fibroblasts treated with CPT (0.5 microgram/mL). The immunostain for bcl-2 was done 12 hours after a microinjection of antisense oligomer to bcl-2 in the nuclei of the cells. RESULTS: On immunocytochemistry, bcl-2 showed no expressions regardless of CPT treatment and microinjection of the antisense oligomer. The expression of cytochrome c was not changed before and after CPT treatment, and bax demonstrated weak or moderate expressions at 36 and 48 hours afte the treatment. There were no expressions at 0, 12, and 24 hours after CPT treatment. On westem blot, bcl-2 exhibited no expressions before and after CPT treatment. Expressions of ctyochrome c and caspase-3 increased after CPT treatment, and expressions of bax decreased 24 hours after CPT treatment followed by a tendency of increased expressions as time went by. CONCLUSIONS: In the CPT-induced apoptosis of mouse 3T3 fibroblasts, CPT induced increased expressions of bax, cytochrome c and caspase-3 with no expressions of bcl-2, which are associated with the apoptosis pathway.

A Study of Apoptosis Induced by Microinjection of Cytochrome c Protein into Mouse 3T3 Fibroblast

BACKGROUND: Microinjectors have been used for cell biology and development, and are useful for the study of cellular morphologic changes with response to the external milieu and intracellularly injected molecules. METHODS: This study was performed to confirm the apoptotic changes induced by intracytoplasmic microinjection of cytochrome c (5 mg/mL) to mouse 3T3 fibroblasts with and without pretreatment of Ac-DEVD-CHO (100 mol/mL), and BSA (bovine serum albumin, 5 mg/mL) as a control, and evaluate the usefulness of microinjection as a method to study apoptosis pathways. RESULTS: Mild focal cytoplasmic fragmentation was seen in the cells microinjected with cytochrome c as early as 10 min after the injection. Apoptotic morphology with apoptotic body formation was observed at 60 min after the injection, and then new apoptotic change of the injected cells was not seen. Cytochrome c-injected cells showed about 31% of apoptotic cells of the total injected cells 50-60 min after the injection. BSA-injected cells did not show apoptosis morphology at 50-60 min after the injections. Caspase-3 inhibitor, Ac-DEVD-CHO-treated cells with cytochrome c microinjection exhibited lower apoptosis indices (average apoptosis index; 11.5+/-8.6%) than non-treated cells of the inhibitor (average apoptosis index; 11.5+/-8.6%). CONCLUSIONS: It was observed that intracellular microinjection of cytochromic c induced apoptosis which was inhibited by Ac-DEVD-CHO, although apoptotic cells were so easily detached that further study could not be performed. However it is thought that microinjection should be a method to study apoptosis and signal transduction with the molecular biological techniques currently available.